Studies in this program over the last five years have shown that angiotensin II (ANGII) is an important growth factor, that it is required for the maintenance of normal function of resistance vessels in the skeletal muscle and brain, and that it is importantly involved in salt sensitive hypertension. We have recently established that ANGII plays a key role in angiogenesis in adult skeletal muscle and have established a model of chronic electrical stimulation of the hind limb musculature in rats that produces a robust and reproducible angiogenesis. In this model, high salt diet or inhibition of ANGII formation blocks "stimulated angiogenesis", but the mechanism by which this occurs, or the pathways that are interacting, have not been established. In Project 5, we hypothesize that ANGII, which is produced locally in blood vessels of skeletal muscle in response to electrical stimulation, interacts with nitric oxide, and 20-HETE, to alter the balance between antiangiogenic (homeostatic) and angiogenic mechanisms resulting in an increase in microvessel density. In this project we will take advantage of several genetically manipulated rat strains that will help us to better dissect out the roles of ANGII and sodium intake in the regulation and maintenance of the microcirculation. The Dahl S rat (SS/Mcw) is a model of salt sensitive hypertension with low renin levels on both high and low salt diets. This rat does not undergo angiogenesis in response to the electrical stimulation protocol used in this application. The consomic rat model (SS.BN13) in which chromosome 13 (the chromosome containing the renin gene) has been substituted from the BN/Mcw rat strain into the isogenic background of the parental SS/Mcw rat is 98% identical to the SS/Mcw strain, yet it is normotensive and has restored renin and angiogenic responses making it ideal for the studies in this project. Aim 1 will use these animals to determine the importance of locally produced ANGII in angiogenesis induced by electrical stimulation using our highly specific and sensitive extraction and assay procedures for the measurement of ANGII in tissue. The unique strains of inbred rats will allow us to separate a potential causative from a permissive role for ANGII. Aim 2 will take advantage of the availability of a series of well-characterized inhibitors and assay systems to investigate the interactions between the renin-angiotensin system, nitric oxide, and 20-HETE production in skeletal muscle angiogenesis in vivo. Additional studies in a unique skeletal muscle, microvascular, endothelial cell culture model will allow confirmation of results obtained in vivo. Aim 3 focuses on mechanisms involved in the inhibition of angiogenesis by high salt diet and will test two specific hypotheses regarding the regulation of production ANGII and its receptors in skeletal muscle. In these studies the role of blood pressure and salt intake will be separated using our strains of inbred rats in which salt sensitivity of blood pressure and angiogenesis are not always related. Regulation of ANGII receptor subtypes will be studied using our techniques for hand dissection and isolation of microvessels followed by quantitative real time RT-PCR and Western blotting techniques. Taken together, these studies will define the role of the renin-angiotensin system in the regulation of angiogenesis in skeletal muscle and may provide insight into the clinical benefit of inhibition of the RAS in situations where angiogenesis is counterproductive.